C). Similarly to observations on S. Typhimurium,
the pathogen loads in the feces of GF mice, or
of GF mice previously reconstituted with the
microbiota of 4-day-old mice and then orally
infected with C. rodentium, were reduced by 4
to 5 logs after administration of Clostridia (Fig.
3, D and E). No loss of C. rodentium was seen
when a mixture of Bacteroides species was given
to GF mice reconstituted with the microbiota
of neonatal mice (Fig. 3, D and E).

We next asked whether host immunity plays
a role in Clostridia-mediated colonization resistance against S. Typhimurium infection in the
intestine. The microbiota from 4-day-old mice
were transferred to wild-type, mutant GF mice
deficient in Myd88/Trif, two essential adaptors
for signaling via the Toll-like/interleukin-1 (IL-1)/
IL-18 receptor family, or Rag1−/− GF mice that
are devoid of B and T cells. All these reconstituted GF mice exhibited unimpaired colonization resistance against S. Typhimurium infection
upon intragastric administration of Clostridia
compared with GF mice that were not gavaged
with Clostridia (fig. S5). Thus, colonization resistance against S. Typhimurium in the intestine
does not require host stimulation via innate
MyD88/Trif-regulated pathways or adaptive immunity. Certain antimicrobial proteins, including
regenerating islet–derived 3 beta (Reg3b) and IL-
22–induced Reg3g, have been associated with
colonization resistance to pathogens in some systems (13). Notably, the expression of Reg3b, Reg3g,
and Il6, but not Muc2 or Tnfa, was higher in
the cecum of GF mice colonized with the adult
microbiota than in GF mice colonized with the
microbiota of 4-day-old mice (fig. S6). However,
the expression of Reg3b and Reg3g was reduced
in Myd88−/−Ticam−/− GF mice colonized with the
adult microbiota (fig. S6). Likewise, the expression
of Il22, a cytokine involved in the regulation of
intestinal barrier function and Reg3g (14), was
reduced in the intestine of GF mice colonized with
the microbiota of 4-day-old mice compared with
that of adult mice (fig. S6). However, treatment
with a neutralizing antibody to IL-22 to inhibit
IL-22–mediated protection (15) neither affected
S. Typhimurium loads in fecal and cecal contents
nor influenced colon length in infected GF mice
reconstituted with the microbiota of adult mice
(fig. S7).

To determine whether Clostridia protected
neonatal mice from pathogen challenge, 10-day-old
mice were gavaged with the Clostridia consortium
or left untreated and then intragastrically infected
with the S. Typhimurium DspiA mutant. Notably, ~50% of the neonatal mice inoculated with
S. Typhimurium succumbed to infection, whereas
>90% of the neonatal mice previously gavaged
with Clostridia survived (Fig. 3F). Collectively,
these results indicate that Clostridia mediate colonization resistance against S. Typhimurium and
C. rodentium via a mechanism that is independent of Myd88, Trif, B, and T cells. Furthermore,
administration of Clostridia protects neonatal mice
from mortality induced by pathogen challenge.

With the exception of a few LachnospiraceaeOTUs, which are present in the microbiota of12-day-old mice, taxa in the order of Clostridialesare absent from the microbiota of 4-day-old and12-day-old mice but robustly colonize the intes-tine between days 12 and 16 of neonatal life, thetime frame associated with the acquisition ofcolonization resistance against pathogens. To as-sess whether neonatal bacteria promote the colo-nization of Clostridia species, GF mice were firstcolonized with the microbiota from 4-day-oldmice, and 7 days later they were gavaged withthe Clostridia consortium. The abundance ofClostridium IV and XIVa clusters, which con-stitute the predominant Clostridia in the con-sortium assessed by quantitative polymerasechain reaction (qPCR), was low after intragastricgavage to GF mice (Fig. 4A and fig. S8A). In thepresence of the 4-day-old neonatal microbiota,the intestinal colonization of Clostridia increasedby ~6 logs (Fig. 4A and fig. S8A). Thus, coloniza-tion of Clostridia is reduced in the absence ofneonatal bacteria. However, if GF mice werereconstituted with the microbiota of 4-day-oldmice, then subsequent intragastric administra-tion of Clostridia induced robust colonizationresistance against S. Typhimurium (fig. S9). Like-wise, preinoculation of GF mice with Lactobacillusmurinus or E. coli, species that are present in4- and 12-day-old neonatal microbiota, respec-tively, or with Bacteroides acidifaciens whosecolonization coincides with robust acquisitionof Clostridiales in the microbiota of 16-day-oldmice, enhanced the colonization of Clostridia by5 to 6 logs (Fig. 4B and fig. S8B).To assess whether bacteria-derived metabo-lites regulate intestinal colonization by Clostridia,we performed unbiased capillary electrophoresis–time-of-flight mass spectrometry (CE-TOFMS)–based metabolome analysis of the cecal contentsof GF mice and GF mice colonized with dom-inant bacterial species present in the ceca ofneonatal and adult mice. The metabolome anal-ysis revealed that amounts of succinate were verylow in the cecal contents of GF mice. Succinatelevels were also low in GF mice reconstitutedwith Clostridia, slightly higher in GF mice col-onized with E. coli, and significantly elevated inGF colonized with Bacteroides when comparedwith GF mice (Fig. 4, C and D). Succinate levelswere increased in GF mice reconstituted with themicrobiota of 12- and 16-day-old mice, but not inthose given microbiota of 4-day-old mice or givenlactobacilli (Fig. 4, D and E), indicating that anincrease in succinate levels is not required forClostridia colonization. Administration of succi-nate, but not acetate or lactate, in drinking waterdid, however, enhance colonization of Clostridiabelonging to the dominant IV and XIVa clustersby 4 to 5 logs (Fig. 4F and fig. S8C). Consistentwith these results, succinate in the drinking waterreduced the intestinal loads of S. TyphimuriumDspiA by ~100-fold in GF mice given theClostridia consortium by gavage (Fig. 4G). Aero-bic and facultative anaerobic bacteria have beensuggested to consume oxygen in the distal intes-tine, which then promotes the colonization ofstrict anaerobes (16). We found that succinateadministration did reduce the concentration ofoxygen in the intestine of GF mice (Fig. 4H).Together, these results indicate that the neo-natal microbiota contribute to the acquisitionof protective Clostridia before weaning and is acritical event that prevents the growth of entericpathogens in the gut early in life.

The authors thank L. Haynes for animal husbandry, D. Peterson for
mouse strains, Genentech for antibody to IL-22, G. Chen and
M. Zeng for critical reading of the manuscript, and the
Germ-Free Animal Core and the Host Microbiome Initiative
at the University of Michigan Medical School for support.

This work was supported by NIH grants DK095782 and DK091191
(G.N.) and AI106302 (C.R.N.); a Career Development
Award from the Crohn’s and Colitis Foundation of America
(Y.-G.K.); fellowships from the Japanese Society for the
Promotion of Science, Kanae Foundation for the Promotion of
Medical Science, and Mishima Kaiun Memorial Foundation
(K.S.); NIH training grant T32DK094775 (J.M.P.); and Grant-in-Aid for
Scientific Research on Innovative Areas “Stem Cell Aging and
Disease” from the Ministry of Education, Culture, Sports, Science and
Technology (15H01522) and the Japan Science and Technology
Agency PRESTO (S.F.). All data and code to understand and assess
the conclusions of this research are available in the main text,
supplementary material, and from the following repositories:
Microbiota data files are available at www.ncbi.nlm.nih.gov/bioproject/
378417 in the National Center for Biotechnology Information (NCBI)
Sequence Read Archive under BioProject PRJNA378417 (SRA:
SRP101509), and the metabolomics data is available from the
Metabolomics Workbench at www.metabolomicsworkbench.org/data/
DRCCMetadata.php?Mode=Study&StudyID=ST000570&StudyType=
MS&Result Type=1 (accession number ST000570; Project
PR000418). Clostridia consortium from the University of Chicago
(CL-UC) is available from the University of Chicago under a material
transfer agreement with the University of Chicago. Y.-G.K. and
G.N. conceived and designed experiments. Y.-G.K. and K.S. conducted
most of the experiments, with help from S.-U.S., J.M.P., N.A.P., M.H.,
and X.L. S.F. performed metabolome analysis. T.M.S., E.C.M., T.D. W.,
and C.R.N. provided advice, discussion, and critical materials. T.F.,
A. T.S., and J.M.P. provided critical materials. Y.-G.K., K.S., S.F., N.I.,
and G.N. analyzed the data. Y.-G.K., K.S., and G.N. wrote the
manuscript, with contributions from all authors. C.R.N. is president and
cofounder of ClostraBio, Inc., a company developing microbiome-modulating therapeutics for the treatment of food allergies. Y.-G.K.,
C.R.N., and G.N. are coinventors on patent application 62/442,527,
submitted by the University of Chicago and the University of Michigan,
which is related to the treatment of enteric disease with Clostridia.